Vanessa Lavallard

ADCY5 Couples Glucose to Insulin Secretion in Human Islets

Single nucleotide polymorphisms (SNPs) within the ADCY5 gene, encoding adenylate cyclase 5, are associated with elevated fasting glucose and increased type 2 diabetes (T2D) risk. Despite this, the mechanisms underlying the effects of these polymorphic variants at the level of pancreatic β-cells remain unclear. Here, we show firstly that ADCY5 mRNA expression in islets is lowered by the possession of risk alleles at rs11708067. Next, we demonstrate that ADCY5 is indispensable for coupling glucose, but not GLP-1, to insulin secretion in human islets. Assessed by in situ imaging of recombinant probes, ADCY5 silencing impaired glucose-induced cAMP increases and blocked glucose metabolism toward ATP at concentrations of the sugar >8 mmol/L. However, calcium transient generation and functional connectivity between individual human β-cells were sharply inhibited at all glucose concentrations tested, implying additional, metabolism-independent roles for ADCY5. In contrast, calcium rises were unaffected in ADCY5-depleted islets exposed to GLP-1. Alterations in β-cell ADCY5 expression and impaired glucose signaling thus provide a likely route through which ADCY5 gene polymorphisms influence fasting glucose levels and T2D risk, while exerting more minor effects on incretin action.

Postprandial macrophage-derived IL-1β stimulates insulin, and both synergistically promote glucose disposal and inflammation

The deleterious effect of chronic activation of the IL-1β system on type 2 diabetes and other metabolic diseases is well documented. However, a possible physiological role for IL-1β in glucose metabolism has remained unexplored. Here we found that feeding induced a physiological increase in the number of peritoneal macrophages that secreted IL-1β, in a glucose-dependent manner. Subsequently, IL-1β contributed to the postprandial stimulation of insulin secretion. Accordingly, lack of endogenous IL-1β signaling in mice during refeeding and obesity diminished the concentration of insulin in plasma. IL-1β and insulin increased the uptake of glucose into macrophages, and insulin reinforced a pro-inflammatory pattern via the insulin receptor, glucose metabolism, production of reactive oxygen species, and secretion of IL-1β mediated by the NLRP3 inflammasome. Postprandial inflammation might be limited by normalization of glycemia, since it was prevented by inhibition of the sodium–glucose cotransporter SGLT2. Our findings identify a physiological role for IL-1β and insulin in the regulation of both metabolism and immunity.

Cadherin Engagement Improves Insulin Secretion of Single Human β-Cells

The aim of this study was to assess whether cadherin-mediated adhesion of human islet cells was affected by insulin secretagogues and explore the role of cadherins in the secretory activity of β-cells. Experiments were carried out with single islet cells adherent to chimeric proteins made of functional E-, N-, or P-cadherin ectodomains fused to the Fc fragment of immunoglobulin (E-cad/Fc, N-cad/Fc, and P-cad/Fc) and immobilized on an inert substrate. We observed that cadherin expression in islet cells was not affected by insulin secretagogues. Adhesion tests showed that islet cells attached to N-cad/Fc and E-cad/Fc acquired, in a time- and secretagogue-dependent manner, a spreading form that was inhibited by blocking cadherin antibodies. By reverse hemolytic plaque assay, we showed that glucose-stimulated insulin secretion of single β-cells was increased by N-cad/Fc and E-cad/Fc adhesion compared with control. In the presence of E-cad/Fc and after glucose stimulation, we showed that total insulin secretion was six times higher in spreading β-cells compared with round β-cells. Furthermore, cadherin-mediated adhesion induced an asymmetric distribution of cortical actin in β-cells. Our results demonstrate that adhesion of β-cells to E- and N-cadherins is regulated by insulin secretagogues and that E- and N-cadherin engagement promotes stimulated insulin secretion.

Aberrant Accumulation of the Diabetes Autoantigen GAD65 in Golgi Membranes in Conditions of ER Stress and Autoimmunity.

Pancreatic islet β-cells are particularly susceptible to endoplasmic reticulum (ER) stress, which is implicated in β-cell dysfunction and loss during the pathogenesis of type 1 diabetes (T1D). The peripheral membrane protein GAD65 is an autoantigen in human T1D. GAD65 synthesizes γ-aminobutyric acid, an important autocrine and paracrine signaling molecule and a survival factor in islets. We show that ER stress in primary β-cells perturbs the palmitoylation cycle controlling GAD65 endomembrane distribution, resulting in aberrant accumulation of the palmitoylated form in trans-Golgi membranes. The palmitoylated form has heightened immunogenicity, exhibiting increased uptake by antigen-presenting cells and T-cell stimulation compared with the nonpalmitoylated form. Similar accumulation of GAD65 in Golgi membranes is observed in human β-cells in pancreatic sections from GAD65 autoantibody-positive individuals who have not yet progressed to clinical onset of T1D and from patients with T1D with residual β-cell mass and ongoing T-cell infiltration of islets. We propose that aberrant accumulation of immunogenic GAD65 in Golgi membranes facilitates inappropriate presentation to the immune system after release from stressed and/or damaged β-cells, triggering autoimmunity.

Cell rearrangement in transplanted human islets

The major feature of the human pancreatic islet architecture is the organization of endocrine cells into clusters comprising central β cells and peripheral α cells surrounded by vasculature. To have an insight into the mechanisms that govern this unique islet architecture, islet cells were isolated, and reaggregation of α and β cells into islet‐like structures (pseudoislets) after culture or transplantation into mice was studied by immunohistology. The pseudoislets formed in culture displayed an unusual cell arrangement, contrasting with the transplanted pseudoislets, which exhibited a cell arrangement similar to that observed in native pancreatic islet subunits. The pattern of revascularization and the distribution of extracellular matrix around transplanted pseudoislets were alike to those observed in native pancreatic islets. This organization of transplanted pseudoislets occurred also when revascularization was abolished by treating mice with an anti‐VEGF antibody, but not when contact with extracellular matrix was prevented by encapsulation of pseudoislets within alginate hydrogel. These results indicate that the maintenance of islet cell arrangement is dependent on in vivo features such as extracellular matrix but independent of vascularization.—Lavallard, V., Armanet, M., Parnaud, G., Meyer, J., Barbieux, C., Montanari, E., Meier, R., Morel, P., Berney, T., Bosco, D. Cell rearrangement in transplanted human islets. FASEB J. 30, 748–760 (2016). www.fasebj.org

Human Amniotic Epithelial Cells Integrated Into the Islet Heterospheroids Enhance Insulin Secretion and Protect Islet Cells from Hypoxic Injury

Background Recent advancements in tissue engineering shows that generating multicellular spheroids by combining different cell types can enhance their regenerative capacity. Human amniotic epithelial cells (hAECs) gained great interest in regenerative medicine due to their availability, safety, regenerative, immunomodulatory and anti-inflammatory properties. The aim of this study was to determine whether combination of hAECs with islet cells in the same spheroid would further improve islet cell survival and function in vitro under normoxic and hypoxic conditions. Methods Functional Islet spheroids were generated on 3D agarose-patterned microwells. To form homospheroids dispersed rat islet cells (ICs) and hAECs (128,000 cells/mold and 500cell/spheroid) were seeded alone. Heterospheroids were formed by mixing ICs and hAECs at ratio of 1:1. Engineered islet homo- and heterospheroids were cultured under normoxic and hypoxic conditions for 16 h. For all conditions, cell viability, GSIS, total islet cellular insulin content were detected. Hypoxia-induced changes in gene expression were assessed by real time PCR analysis. Next we considered possible molecular mechanisms behind the beneficial effect of hAECs on islet cell function. For this purpose, the mRNA expression levels of Hif-1&agr;, Casp3, Casp8, Casp9 and Bcl2 were determined. Results Confocal laser scanning microscopy showed uniform distribution of islet cells and hAECs throughout of hybrid spheroids, without evidence of cell loss. Quantifications of the insulin positive area ratios to nuclei, showed that heterospheroids expressed more insulin compared to homospheroids. Moreover, islet heterospheroids expressed significantly more E-cadherin, key cell-to-cell adhesion molecule then homospheroids. The stimulation index of the heterospheroids was significantly higher than those of homospheroids. Exposure to hypoxia rapidly caused fragmentation of homospheroids and augmented cell membrane permeability. In contrary considerably less dead signals were observed within heterospheroids. As anticipated, glucose responsiveness of homospheroids was seriously impaired; the average glucose stimulation index of the heterospheroids was significantly higher than that of the homospheroids (1.7±0.4 vs 0.9±0.5, p < 0.05). This was correlated with downregulation of apoptotic genes Casp3, Casp8 and Casp9 and 2 fold upregulation of antiapoptotic gene Bcl2. Conclusions These data indicate that incorporation of hAECs into the islet heterospheroids improves the secretory function and viability of islet cells both in conventional culture and in hypoxic conditions.

Shielding Human Islets with Human Amniotic Epithelial Cells Enhances Islet Engraftment and Revascularization

Background Hypoxia is a main cause of considerable islet loss during first days after intraportal transplantation. The aim of this study was to investigate whether shielding of human islets (HI) with human amniotic epithelial cells (hAECs), which are known to possess immunomodulatory, anti-inflammatory and regenerative properties could improve islet engraftment and survival. Methods Shielded islets were generated on microwells by mixing HI and hAECs at ratio of 1:800 (800 hAECs per HI). Next, 1200 shielded or neat islets, were transplanted under the kidney capsule of diabetic SCID mice. The ability of hAECs to adhere to human islets was analyzed by confocal microscopy. Islet function was assessed by dynamic insulin release in response to glucose in vitro. Blood glucose and weight were monitored regularly. Intravenous glucose tolerance test was performed 1 month after transplantation. Graft morphology and vascularisation were evaluated by immunohistochemistry. Results HI shielded with hAECs had greater cellular insulin content and increased glucose-stimulated insulin secretion. Transplantation of shielded islets resulted in considerably earlier normoglycemia and vascularization, improved glucose tolerance, and increased insulin content. Conclusion Co-transplantation of islets with hAECs had a profound impact on the remodelling process, maintaining islet organisation and improving islet revascularisation. Moreover, hAECs improved the capacity of islets to reverse hyperglycaemia.

Islet Heterospheroids Generated from Islet Cells and Amniotic Epithelial Cells Reverse Diabetes After Marginal Mass Transplantation in a Murine Model

Background Human amniotic epithelial cells (hAECc) derived from placental tissue are widely available and possess immunomodulatory, anti-inflammatory and regenerative properties. In this study we have generated islet heterospheroids composed of hAECs and dispersed islet cells (ICs) aiming to improve viability, engraftment and vascularization of the transplanted spheroids. Methods Functional Islet spheroids were generated on 3D agarose-patterned microwells. To form homospheroids dispersed rat islet cells (ICs) and hAECs (128,000 cells/mold and 500cell/spheroid) were seeded alone. Heterospheroids were formed by mixing ICs and hAECs at ratio of 1:1 (128,000 cells/mold and 500cell/spheroid). Marginal mass (150 IEQ) of islet heterospheroids (islet + AEC group), islet homospheroids (islet-only group) or hAEC spheroids (hAEC alone group) was transplanted under the kidney capsule of diabetic SCID mice. Blood glucose levels were monitored daily and IPGTTs were carried out. Grafts and serum were harvested at 1, 2, 6 and 12 weeks after transplantation to assess outcome. Results Mice transplanted with islet heterospheroids exhibited enhanced glycemic control as measured by glucose tolerance, serum insulin/c-peptide level and diabetes reversal rate, compared with mice in islet alone group. The cumulative percentage of animals reaching normoglycemia was 74% in the islet+hAEC group versus 26% in the islets-alone group. The median time to reverse hyperglycaemia for islet+hAEC grafts was 5 ± 0.9 days and 30 ± 7 days for islet-alone recipients (p < 0.0001, n = 26). Between groups, the morphology of islet grafts showed significant differences in size and composition of grafted endocrine tissues. A two-fold increase in graft revascularization was seen in islet + hAEC grafts, which was mainly attributed to stimulating vascular endothelial growth factor-A (VEGF-A) production. The rapid revascularization led to improved graft perfusion and recovery from hypoxia. Conclusion These data indicate that hAECs may have a significant potential to protect islet cells and may be employed to improve islet cell survival and function prior to transplantation. Hence, hAEC-enriched pseudoislets may represent a novel approach to increase the success rate of islet transplantation.

Effects of remote ischemic preconditioning on intraportal islet transplantation in a rat model

Remote ischaemic preconditioning (RIPC), which is the intermittent interruption of blood flow to a site distant from the target organ, is known to improve solid organ resistance to ischaemia‐reperfusion injury. This procedure could be of interest in islet transplantation to mitigate hypoxia‐related loss of islet mass after isolation and transplantation. Islets isolated from control or RIPC donors were analyzed for yield, metabolic activity, gene expression and high mobility group box‐1 (HMGB1) content. Syngeneic marginal mass transplantation was performed in four streptozotocin‐induced diabetic groups: control, RIPC in donor only, RIPC in recipient only, and RIPC in donor and recipient. Islets isolated from RIPC donors had an increased yield of 20% after 24 h of culture compared to control donors (P = 0.007), linked to less cell death (P = 0.08), decreased expression of hypoxia‐related genes (Hif1a P = 0.04; IRP94 P = 0.008), and increased intra‐cellular (P = 0.04) and nuclear HMGB1. The use of RIPC in recipients only did not allow for reversal of diabetes, with increased serum HMGB1 at day 1; the three other groups demonstrated significantly better outcomes. Performing RIPC in the donors increases islet yield and resistance to hypoxia. Validation is needed, but this strategy could help to decrease the number of donors per islet recipient.

Combined Pancreatic Islet-Lung-Liver Transplantation in a Pediatric Patient with Cystic Fibrosis-Related Diabetes

Background: Cystic fibrosis-related diabetes (CFRD) is the most frequent extrapulmonary complication of cystic fibrosis (CF). Methods: We report the first combined pancreatic islet-lung-liver transplantation in a 14-year-old adolescent. CFTR was analyzed by Sanger sequencing. Further genes were analyzed by high-throughput sequencing. Results: The patient was diagnosed with CF at the age of 14 months. Nine years later, after diagnosis of CFRD, the patient’s BMI and lung function began to decline. Bilateral lung transplantation with simultaneous liver transplantation was performed at the age of 14.5 years. The first islet transplantation (IT) was carried out 10 days later. Six months later, C-peptide secretion after arginine stimulation showed peak values of 371 pmol/L (vs. 569 pmol/L before IT) and insulin doses had slightly increased (1.40 vs. 1.11 units/kg/day before IT). A second IT was performed at the age of 15 years, a third at 16 years. Two years after the first IT, arginine-stimulated C-peptide secretion increased to 2,956 pmol/L and insulin doses could be reduced to 0.82 units/kg/day. HbA1c decreased from 7.3% (57.4 mmol/mol) to 5.9% (41.0 mmol/mol). Conclusion: IT following lung and liver transplantation, with injection of islets into a transplanted organ, is feasible. It improves C-peptide secretion, decreases insulin needs, and lowers HbA1c.